POS1422 SINGLE-CELL RNA SEQUENCING OF HAEMATOPOIETIC STEM/PROGENITOR CELLS IN HUMAN SLE: EVIDENCE FOR A CHRONIC IFN EXPOSURE AFFECTING EARLY HAEMATOPOIETIC PROGENITORS IN SLE, AND DRIVING THEIR PROLIFERATION AND MYELOID SKEWING
Authors: A. Filia, M. Grigoriou, A. Banos, I. Mitroulis, I. Kokkinopoulos, D. Boumpas
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> All immune cells that contribute to the pathogenesis of systemic lupus erythematosus (SLE) derive from adult haematopoietic stem and progenitor cells (HSPCs) within the bone marrow (BM). We reasoned that the fundamental abnormalities in the disease can be traced back to HSPCs. <h3>Objectives</h3> I) To delineate human haematopoietic CD34+ progenitor subpopulations that may be involved in the pathogenesis of the disease and II) to identify deregulated pathways in each subpopulation in SLE as compared to healthy controls. <h3>Methods</h3> BM samples from 6 SLE patients and 5 healthy controls and 2 umbilical cord blood (CB) samples were used. CD34+ cells were isolated from BM and CB samples and single cell capture and RNA extraction were performed on Fludigm C1 IFC. Libraries were prepared and 75bp or 150bp paired-end sequencing was performed. Cells were excluded if total reads were &lt;25,000, alignment rate was &lt;50%, the number of detected genes (at least 1 count) was &lt;200 and &gt;6,000 and the percentage of reads mapping to mitochondrial genes was &gt;20%. The Seurat and harmony R packages were used for normalization, marker identification and graph-based clustering. Pseudobulk differential expression (DE) analysis was performed using edgeR R package. Enrichment analysis was performed using EnrichR. <h3>Results</h3> A total of 426 out of 836 cells and 24,473 genes were used in the analysis. There was a statistically significant difference in the total number of detected genes between the three sample types (Kruskal-Wallis chi-squared=14.9, p&lt;0.001) with CB samples expressing more genes compared to the other two sample types. The top 3,000 highly variable genes across cells were identified and loaded into a PCA and the top 10 principal components were provided as the input for the UMAP visualization which resulted in seven distinct clusters of cell types (Figure 1). Mutually exclusive markers, which were characteristic of each cell type were identified. Cluster 0 cells (T-like progenitors) uniquely expressed IL32 and CD3E, whereas cluster 1 (Myeloid-1 progenitors) uniquely expressed MMP8. Of note, there was a significantly lower proportion of SLE BM B-like progenitor cells (3.5% of total SLE BM cells) and SLE BM HSCs-3, characterized by primitive progenitors with low expression of the cell cycle gene MKI67 (4% of total SLE BM cells) compared to 10% B-like progenitors and 21% HSCs-3 of total healthy BM cells respectively. Following DE analysis between SLE and healthy BM there were statistically significant differences (FDR&lt;0.1) in Myeloid-2 progenitors, with differentially expressed genes (DEGs) enriched for TNF signaling and DNA repair pathways, and in HSCs-1, with DEGs enriched for IFN alpha-beta signaling and heme metabolism. Interestingly, we observed an upregulation in the expression of IFN inducible genes, such as IFI44, IFI44L, in SLE compared to healthy in all three HSC subpopulations. <h3>Conclusion</h3> Single-cell RNA sequencing analysis from CD34+ progenitor cells from patients with SLE revealed both quantitative- as evidenced by decreased numbers of non-proliferating early progenitors, and qualitative differences- characterized by an IFN signature, which is known to drive loss of function and attrition of HSPCs. Chronic IFN exposure affects early hematopoietic progenitors in SLE, driving their proliferation and myeloid skewing and may account for the immune abberancies and the cytopenias in SLE. <h3> Acknowledgements</h3> The European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement no.742390) <h3>Disclosure of Interests</h3> None Declared.